The Contractor currently provides a high-throughput assay platform that rapidly pinpoints compound mechanism of action and toxicity liabilities through its unique ability to monitor multiple facets of previously inaccessible biology in live cells in real-time. Through this SBIR Phase II contract, the Contractor's existing platform will be applied to the development of a novel live-cell multiplexed fluorescent assay that reports an aggregate of at least 10 distinct readouts of cellular stress and organelle homeostasis per well. A panel of multicolor cell stress reporters will be generated and stably integrated into cell lines relevant for compound toxicity evaluation. The resulting panel of multicolor stable reporter cell lines will be used as the foundation for our multiplexed assay. Our assay will be validated by testing its response to tool compounds known to induce cellular stress. Our assay's suitability for high-throughput screening will be evaluated by running a 10,000-sample pilot screen, followed by scaling to at least 100,000 samples/day throughput in 384-well plate format with full assay automation. The Contractor's proposed assay will benefit public health by reducing the time and cost to pinpoint toxicities of pharmaceutical and environmental chemicals, thus improving our ability to identify beneficial therapeutics, and to proactively manage harmful environmental toxins.